Preparation of live vaccines

ABSTRACT

Described is a method for the generation of a live vaccine containing stable bacteria carrying at least three attenuating mutations and a vaccine containing bacteria obtained by said method.

The present application is a national phase entry under 35 U.S.C. § 371 of International Patent Application PCT/EP2013/068373, filed on Sep. 5, 2013 and published in English as International Patent Publication WO2014/037445 A1 on Mar. 13, 2014, which claims benefit of priority to European Patent Application Ser. No. 12008206.0, filed Dec. 7, 2012; all of which are incorporated by reference in their entirety.

The present invention provides a method for the generation of a live vaccine containing stable bacteria carrying at least three attenuating mutations and a vaccine containing bacteria obtained by said method.

Many of the live bacterial vaccines comprise attenuated bacteria that have been manipulated by biomolecular techniques. Unfortunately, most of these vaccines are considered as being insufficient to comply with the requirements of practice for the following reasons:

-   -   (a) The production is complex and time consuming, the degree of         attenuation cannot be controlled and, accordingly, adaptation to         the susceptibility of the host is often unsatisfactory.     -   (b) Methods of testing (clinical trials) requested by the         legislative authority are also elaborate.     -   (c) The population to be vaccinated is limited.

By contrast, mutants attenuated by metabolic drift (MD) are characterized by the following advantages:

-   -   (a) Costs for preparation are low and the degree of attenuation         via the desired selection of an increased generation time and,         thus, reduced colony size, respectively, is, in principle,         almost arbitrary.     -   (b) When using stable specific vaccine strains having three         attenuated MD mutations for vaccination of farm animals,         elaborate methods of testing are not required.     -   (c) Even smaller lot sizes will pay off.

As regards key data of the evolutionary principle of MD attenuation the following should be stressed:

-   -   (a) The interplay of pathogenic agent versus host is based on         mutual tolerance. Highly susceptible hosts survive as single         individuals when accidentally infected by an attenuated mutant         of a highly virulent pathogenic agent. The host population         rejuvenates via the few surviving individuals. The pathogenic         agent proliferates as adapted attenuated strain. Myxomatosis is         a typical example of such process. Conclusion: Bacterial         populations (as well as populations of fungi and viruses) always         contain gradually attenuated mutants, inter alia so-called MD         mutants.     -   (b) MD mutants represent clones having mutations in metabolic         compartments per definitionem resulting in dysfunction (i.e.         attenuation=fitness cost). As a consequence, gradually reduced         colony sizes (depending on the clone) compared to the wild         strain can be found. Normally, these mutants are eliminated by         the immuno competent host or, alternatively, are overgrown by         the adapted normal flora.     -   (c) The reduced colony sizes of the MD mutants inversely         correlate with the (prolonged) generation time and the         (increasing) degree of attenuation.     -   (d) The convincing efficacy of MD attenuated test vaccines and         vaccines has been proven.

MD mutants can be selected and isolated as:

-   -   (a) spontaneous MD antibiotic resistence (MD “res”) clones of,         e.g., streptomycin, rifampicin, fosfomycin, fusidic acid,         nalidixic acid. These clones can be isolated with a frequency of         more than 1% in relation to the virulent resistant clones. MD         “res” and virulent resistant clones result from different         mutations. Accordingly, MD “res” and attenuation can be regarded         as a functional entity.     -   (b) Increased environmental stress tolerance (iet) mutants which         indirectly accumulate in the “dying off” culture.     -   (c) streptomycin independent (Sm-id) suppressor mutants derived         from streptomycin dependent (Smd) clones. These two marker         mutants consist of a broad spectrum of clones characterized by         clone specifically graduated reduced colony sizes and increasing         degrees of attenuation, respectively, from almost wild type         virulence to over-attenuation (mini colonies). Generally,         ribosomal mutations increase the normal misreading         (mistranslation) more or less and the exclusive suppressor         mutation also causes attenuation.

For immunization of, e.g., populations of chicken with live Salmonella and Campylobacter vaccines the interruption of the chain of infection to human beings and, as a consequence, the reduction of human enteritides is the primary goal. Normally, chicks tolerate facultative pathogenic Salmonella (and generally even Campylobacter) without showing any clinical symptoms. Thus, the low virulence of these wild strains for chicks requires vaccine strains showing a moderate degree of attenuation ensuring on the one hand immunogenicity for chicks but excluding on the other hand a hazard for human beings.

One criterion of the efficacy of vaccine strains is, e.g., the verifiable reduction of the degree of colonization after challenge. MD attenuated live vaccines expressing all components of the bacteria (e.g., outer membrane proteins) can be regarded as a practice oriented option, even as regards Campylobacter.

Thus, it is possible to develop effective vaccines for facultative pathogenic bacteria such as Salmonella and Campylobacter, provided over-attenuation is avoided by adjusting to a low or moderate degree of attenuation. In other words, the reduction of colony size as attenuation equivalent should not fall below about 25% of the colony size of the wild strain. In addition, this condition sine qua must be in line with the safety requirements of the WHO:

stability due to the presence of two independent attenuating mutations. The reversion rate per marker is about 10⁻⁸. However, there is a need for developing vaccine strains showing even higher stability, i.e., lower reversion rates, but not over-attenuation. Unfortunately, the introduction of additional mutations to increase the safety of a live vaccine usually leads to on excess of attenuation thereby rendering the vaccine less effective.

Thus, the technical problem underlying the present invention is to provide improved live vaccine strains characterized by increased stability.

The solution of said technical problem is achieved by providing the embodiments characterized in the claims, i.e., to provide improved live vaccine strains with an increased stability based on at least three mutations, yet avoiding over-attenuation and allowing for the adjustment of the attenuation to a desired level. In fact, during the experiments leading to the present invention it could be shown that by use of the MD attenuation vaccine strains characterized by three (or even more) independent attenuating mutations can be generated showing increased stability and a degree of attenuation that does not exceed the degree of attenuation of vaccine strains having two MD “res” attenuating mutations. In the experiments of the present invention streptomycin was used, however the procedures disclosed in the present invention are not restricted to only this antibiotic. The experiments described below are based on the use of so-called Sm-id clones derived from Smd mutants since these “double marker” mutants (comprising clones showing all degrees of attenuation—from wild strain-like colonies to mini colonies) allow for the generation of vaccine strains showing a degree of attenuation corresponding to the degree of attenuation of MD “res” single marker strains. Strains of the present invention (Sm-id/MD “res”) show an increased stability of about 10⁻²⁴.

So far, Sm-id/MD “res” vaccine strains of Salmonella and Campylobacter have not been described in the prior art. In addition, vaccine strains having four, five or even six attenuating mutations generated by the graduated incorporation of two or three Sm-id mutations and an additional MD “res” (A a, A α) marker are novel as well, for streptomycin: Smd 1→Sm-id I→Smd a→Sm-id II→Sm-α→Sm-id III (six attenuating mutations) and Sm-id I/Sm-id II/MD “res” (five attenuating mutations), respectively.

It was found that the particular spectrum of revertants—graduatedly reduced colony sizes—of the Sm-id mutants with combinations of two, four or six markers starts: (a) for Sm-id strains with colony sizes of the wild strain, (b) for Sm-id I/Sm-id II strains with colony sizes of Sm-id I-like colony sizes and (c) for Sm-id I/Sm-id II/Sm-id III constructs the largest colony found only corresponded to Sm-id I/Sm-id II-like clones. Thus, these are not wild strain-like revertants.

In principle, the selection and isolation of Smd mutants as starting strains for the generation of Sm-id clones belongs to the state of the art. About 10% of the normal Sm-resistant mutant colonies of, e.g., Staphyloccoci, Escherichia coli, Bacillus cereus, are Smd clones (as an apparent biological principle). However, this does not apply to Salmonella and Campylobacter. The frequency of the appearance of Smd clones of Salmonella among the colonies of mutants showing resistance is reported to be about 0.1% or the isolation of such strains is only achievable by use of mutagenesis, respectively.

Interestingly, after analysis of about 5000 Sm resistant mutants the present inventors could not find any Smd strains. In addition, it has to be stressed that the spectrum of Sm-id revertants varies depending on the Smd clone, accordingly, several Smd clones are needed as starting mutants. In other words, the common procedure for selection is inapplicable.

Since presumably ribosomal Smd-pathways are an evolutionary principle of adaptation two possible mechanisms were tested:

-   -   (a) Smd-mutants enter the death phase immediately after their         formation. Thus, such mutants can only be isolated from         log-phase cultures, e.g., from ≤18 h/37° C. cultures but not         from ≥48 h/37° CF cultures     -   (b) During status nascendi the Smd clones are fragile, show         delayed growth as mini colonies (which might be overlooked).         These mini colonies adapt to “normal growth” during passaging.

For Campylobacter there are no data available as regards Smd- and Sm-id mutants. In the literature, there are only some hints as regards Sm-resistant mutants: clones showing high resistance could only be obtained by use of multiple passages against increasing concentrations of streptomycin. A one-step Sm-resistance could only be achieved by use of a concentration of 20 μg streptomycin/ml. Experiments of the present inventors for isolating mutants showing Sm-resistance and Smd-mutants using 100 μg streptomycin/ml (by plating of 72 h/39° C. cultures) failed. Surprisingly, it was found that despite significantly increasing amounts of bacteria the viable counts significantly decreased from the 24 h/39° C. to the 72 h/39° C. culture as shown in the following table.

TABLE 1 Campylobacter coli and Campylobacter jejuni: Viable counts on Petri dishes with Caso-agar against the incubation period at 39° C. Campy1obacter 24 h 48 h 72 h C. coli 1 × 10¹⁰ 3 × 10⁹ 7 × 10⁸ C. jejuni 3 × 10¹⁰ 5 × 10⁹ 1 × 10⁹ C. coli Smd 3 × 10⁹  3 × 10⁸ 4 × 10⁷

These results are in line with the observation that cultures of Campylobacter are capable of passing into a viable but not culturable status.

Accordingly, mutants having Sm resistance could only be obtained by plating of ≤24 h/39° C. culture material. However, no Smd mutants could be found among the resistant colonies having normal size or slightly reduced size. But among the very small colonies and mini colonies of Campylobacter (appearing with distinct delay) the majority of the clones could be characterized as being Smd clones.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Examples of Salmonella vaccine strains characterized by three attenuating mutations.

Incubation of the plates was at 37° C. for about 20 hours.

FIG. 1(A) Salmonella infantis (wild strain/Sm-id 4.22/Rif 2 (and Iet 4));

FIG. 1(B) Salmonella virchow (wild strain/Sm-id 9.3/Rif 2);

FIG. 1(C) Salmonella hadar (wild strain/Sm-id 4.1/Sm 2 (and Rif 1);

FIG. 1(D) Salmonella paratyphi B, variant Java 3.2 (wild strain/Sm-id.1.1/Rif 3 (and Rif 2)).

FIG. 2: Examples of Campylobacter vaccine strains characterized by three attenuating mutations. Incubation of the plates was at 39° C. for about 48 and 72 hours, respectively.

FIG. 2(A) Campylobacter coli I (wild strain/Sm-id 5.5/Pho 1 and Pho 2);

FIG. 2(B) Campylobacter coli II (wild strain/Sm-id 18.1/Sm2);

FIG. 2(C) Campylobacter jejuni I (wild strain/Sm-id 2.3/Pho 1 (and Pho 2));

FIG. 2(D) Campylobacter jejuni II (wild strain/Sm-id 2.1/Sm2).

FIG. 3: Examples of Salmonella vaccine constructs characterized by (two), four, five or six attenuating mutations. Incubation of the plates was at 37° C. for about 20 hours.

FIG. 3(A) Salmonella virchow:

wild strain/Sm-id I 1.4/Sm-id II 0.3/Sm 4;

wild strain/Sm-id I 1.4/Sm-id II 0.3/Sm-id III 0.x.

FIG. 3(B) Salmonella Infantis:

wild strain/Sm-id I 4.22/Sm-id II 0.1/Rif 2;

wild strain/Sm-id I 4.22/Sm-id II 0.1/Sm-id III 1.1.

FIG. 4: Examples of Campylobacter vaccine constructs characterized by (two), four, five or six attenuating mutations. Incubation of the plates was at 39° C. for about 48 hours.

FIG. 4(A) Campylobacter coli II wild strain/Sm-id I 18.1/Sm-id II a.1. This strain is characterized in that the second Sm-id a.1 mutation results in an Sm resistance.

FIG. 4(B) Campylobacter coli II wild strain/Sm-id I 17.7/Sm-id II a.1/Phol, wild strain/Sm-id I 17.7/Sm-id II a.1/Sm-id III a.l.

Thus, the present invention provides a method for the generation of a bacterial live vaccine containing stable bacteria carrying at least three (and up to six or seven) attenuating mutations, wherein said method comprises the following steps:

-   -   (a) providing a bacterial strain and growing said strain in the         presence of a first antibiotic, preferably streptomycin;     -   (b) isolating from the strain of (a) such “mini” colonies which         correspond to clones which are dependent on the first         antibiotic;     -   (c) growing a clone of (b) in the absence of the first         antibiotic and isolating attenuated revertants characterized by         a colony size which is ≥50% of the colony size of the wild         strain;     -   (d) growing a clone obtained in step (c) in a medium         supplemented with a second antibiotic that may differ from the         first antibiotic (e.g., an aminoglycoside such as streptomycin,         neomycin, kanamycin, spectinomycin, gentamicin, amikacin, and         tobramycin; rifampicin, fusidic acid, nalidixic acid,         fosfomycin,) having a suitable concentration, preferably an         about tenfold MIC;     -   (e) isolating and serially passaging colonies showing reduced         size (MD A “res”); and     -   (f) isolating clones having the graduated reduction of the         colony size as stable property.

The bacterial strain of step (a) is, preferably, obtained from “wild” virulent strains. These strains can be taken from diseased animals (e.g., chicken). The starting natural strains which are used should have a certain degree of virulence.

The choice of the antibiotic for selecting the mutants of step (a) is guided by reasons of a practical nature. For example, streptomycin is known to lead rapidly to the development of resistant and dependent strains among the micro-organisms.

Thus, in a preferred embodiment of the method of the present invention the antibiotic of step (a) is streptomycin. However, other aminoglycoside antibiotics such as neomycin, kanamycin, spectinomycin, gentamicin, amikacin, and tobramycin, and rifampicin, fusidic acid and nalidixic acid may also be suitable as the antibiotic of step (a).

It is known that resistance to the antibiotic can result from different modification mechanisms. In particular, the genetic modification may affect a chromosome of the bacterium. The chromosomal modification is a rare event which, once carried out, ensures the stability of the acquired properties.

The term “mini colonies” as used herein relates to bacterial colonies characterized by a reduction of size. Preferably, they are characterized by a size of ≤10% of the corresponding wild strain colonies.

The selection of attenuated bacterial strains as a function of growth criteria on media containing an antibiotic is an operation which has been used for various species with the object, notably, of causing the appearance of strains having reduced virulence.

The bacterial strains according to the invention characterized by at least three (and up to six or seven) attenuating mutations are in the first place non-virulent strains selected, from natural virulent strains, for their growth capacity on a medium with a high content of an antibiotic such as streptomycin, and in addition, which can only be developed satisfactorily in the presence of the antibiotic (step (b). For this reason, these strains are said to be dependent on, e.g., streptomycin (Smd mutant).

In the second place, the strains of step (c) are mutants selected from the antibiotic dependent strains and which have the particularity of being able to develop in the absence of streptomycin due to the introduction of a second attenuating mutation or marker. These strains are called Sm-id strains. Preferably, a washing step is carried out between steps (b) and (c). The preferred medium for step (c) is a Salmonella Caso (SC) medium (e.g., for Salmonella) or a Caso medium (e.g., for Campylobacter).

Step (d) allows introducing an additional MD antibiotic resistance (“res”) mutation (as a third attenuating marker). Preferably, the antibiotic is streptomycin, rifampicin or fosfomycin. The concentration of the antibiotic in step (d) can be determined by the person skilled in the art according to routine procedures. Preferably, for Salmonella the concentration of rifampicin, streptomycin (note: most Sm-id mutants are streptomycin sensitive and therefore suited for an additional MD Sm “res” marker) and fosfomycin corresponds to an MIC value of about tenfold, and for fusidic acid to an MIC value of about fourfold. Preferably, for Campylobacter, at least 200 μg fosfomycin/ml and at least 100 μg streptomycin/ml, respectively, are used.

In step (e) the Sm-id/MD antibiotic “res” strains of the previous step characterized by an additional reduced slight colony size are isolated and serially passaged in order to check stability. Preferably, at least 30 serial passages are carried out.

Finally, in step (f) the clones isolated from step (e) having the graduated reduction of the colony size as stable property are provided.

In a preferred embodiment of the method of the invention steps (a) to (c) are at least repeated once for the generation of bacteria carrying at least four attenuating mutations. This method allows to generate new attenuating mutants, e.g., according to the following schemes (shown for Sm):

(a) Smd 1→Sm-id I→Smd a→Sm-id II;

(b) Smd 1→Sm-id I→Smd a→Sm-id II→Smd α→Sm-id III;

(c) Smd 1→Sm-id I→Smd a→Sm-id II→Smd α→Sm-id III→MD antibiotic “res”.

Surprisingly, it was found that strains derived from Sm-id mutants carrying four or even six mutations do not show a higher degree of attenuation, compared to the strains having three attenuating mutations, but an even higher stability.

The choice of the antibiotic for selecting the MD antibiotic “res” mutant strains according to the present invention is guided by reasons of a practical nature and, in principle, any antibiotic capable of inducing metabolic drift (MD) mutations can be used for the purposes of the present invention, e.g., streptomycin (note: most Sm-id mutants are streptomycin sensitive and therefore suited for an additional MD Sm “res” marker), rifampicin, fosfomycin, fusidic acid or nalidixic acid.

The method of the present invention is not restricted to particular bacteria. Besides Salmonella sp. and Campylobacter sp., other bacteria such as Staphylococcus aureus, Escherichia coli, Bacillus cereus (Pseudoanthrax), Yersinia sp. such as Y. pestis, Klebsiella sp., Listeria sp., Aeromonas sp., Shigella sp., Pasteurella/Avibacterium sp., Riemerella sp., Ornithobacterium rhinotracheale, Bordetella sp., and Pseudomonas sp. can also be used for generating bacterial live vaccine containing stable bacteria according to the methods of the present invention.

However, preferred bacteria are Salmonella and/or Campylobacter, especially Salmonella bongori, the S. enterica subspecies enterica, arizonae, diarizonae, salamae, houtenae and indica, preferably S. enterica subspecies enterica such as the following Serovars: Dublin, Gallinarum (biovars Gallinarum and Pullorum), Choleraesuis, Typhisuis, Typhi, Paratyphi A,B,C, Abortusequi, Abortusovis, Abony, Enteritidis, Typhimurium, Copenhagen, Infantis, Virchow, Hadar, Agona, Newport, Anatum, Heidelberg, Panama, Indiana, Saintpaul, Brandenburg, and Campylobacter coli, Campylobacter jejuni, and Campylobacter fetus.

In a preferred embodiment of the method of the present invention in steps (a) and (b) Salmonella mutants are isolated from log phase cultures and as mini-colonies that start appearing after at least or more than 48 h at 37° C. incubation.

In a further preferred embodiment of the method of the present invention in steps (a) and (b) Campylobacter mutants are isolated as mini-colonies that start appearing after at least or more than 72 h at 39° C. incubation.

The present invention also provides alive bacterial strains obtainable by the method of the invention as well as a vaccine comprising alive bacterial strains of the invention and a biologically acceptable carrier. The vaccinating compositions may of course be constituted by means of freshly cultivated bacteria.

Preferably, the vaccine composition of the present invention is freeze-dried.

To administer the vaccinating bacteria, the medium in which they are suspended is not critical. Of course, this medium must not interfere with the good viability of the bacteria that they contain.

The vaccine of the present invention is administered in an amount suitable for immunization of an individual and may additionally contain one or more common auxiliary agents. The employed term “amount suitable for immunization of an individual” comprises any amount of bacteria with which an individual can be immunized. An “amount suitable for immunization of an individual” may be determined using methods known to one skilled in the art. The term “individual” as used herein comprises an individual of any kind. Examples of such individuals are animals (and humans).

The administration of the vaccine preferable is the oral route but also injection may be made at various sites of the individual intramuscularly, subcutaneously, intradermally or in any other form of application. It may also be favourable to carry out one or more “booster injections” having about equal amounts.

The vaccine of the present invention may be prophylactic, that is, the compounds are administered to prevent or delay the development of an infection or colonisation, e.g. an infection/colonisation caused by Salmonella or Campylobacter.

The following strains have been deposited with the German Type Culture Collection (Deutsche Sammlung von 25 Mikrorganismen and Zellkulturen (DSMZ), Braunschweig) on Nov. 27, 2012 under the Budapest Treaty:

Name Accession Number Salmonella enterica ssp. enterica Serovar DSM 26682 Infantis Smid4-22/Rif2 = Campylobacter coli K2848/11 Smid18/Sm2 = DSM 26683 Campylobacter jejuni K2963/12 Smid2.1/Sm2 = DSM 26684

The below examples explain the invention in more detail.

EXAMPLE 1 Materials

(A) Strains

Salmonella enterica subsp. enterica serovar Virchow,

Salmonella enterica subsp. enterica serovar Infantis,

Salmonella enterica subsp. enterica serovar Hadar,

Salmonella paratyphi B (var. L-Tartrat+, formerly Java),

Campylobacter coli, Campylobacter jejuni (provided by Lohmann Animal Health, Cuxhaven, Germany).

(B) Media

1000 ml Campylobacter medium (Caso-medium) contain: 35 g Caso Agar (Sifin), 3 g yeast extract, 3 g casein hydrolysate, 4 g activated carbon, 0.25 g FeSO₄, 0.25 g sodium pyruvate, 5 g agar Kobe (Roth).

1000 ml Salmonella medium (SC-medium) contain: 35 g Caso Agar (Sifin), 3 g yeast extract, 1 g glucose, 5 g agar Kobe (Roth).

(C) Antibiotics

Streptomycin (Sm) (Roth No. 0236.2), fosfomycin (Pho) (Sigma No. P5396), rifampicin (Rif) (Riemser Arzneimittel AG, Fatol Eremfat 600 mg)

(D) MIC values of wild type strains

Strain Streptomycin rifampicin fosfomycin Salmonella enterica 12.5 12.5 n.d. subsp. enterica serovar Virchow Salmonella enterica 12.5 12.5 n.d. subsp. enterica serovar Infantis Salmonella enterica 25 12.5 n.d. subsp. enterica serovar Hadar Salmonella 30 12.5 n.d. paratyphi B (var. L-tartrate+) Campylobacter 1 n.d. 25 coli WS I Campylobacter 1 n.d. 25 coli WS II Campylobacter 2 n.d. 25 jejuni WS I Campylobacter 2 n.d. 25 jejuni WS II n.d.: not determined

EXAMPLE 2 Selection and Isolation, of Smd Mutants

(a) Practice-Orientated Isolation of Smd Mutants of Salmonella

About 10¹⁰ cfu of a 18 h/37° C. culture of Salmonella were plated on a Petri dish containing SC agar supplemented with 500 μg streptomycin/ml. Besides colonies having normal sizes and single colonies having slightly decreased sizes (virulent Sm resistant clones and MD Sm “res” clones) “mini colonies” (predominantly small colony variants=scv) with varying frequencies—depending on the strain—could be detected. After an incubation time of about ≥48 h (at 37° C.) 1 to 2 additional mini colonies (per about 30 colonies having normal sizes and colonies having slightly decreased sizes) could be detected that could not be distinguished from scv. Depending on the frequency of appearance of the scv phenotype 3% to 20% of these mini colonies could be shown to represent Smd mutants.

The calculated frequency of the Smd clones in relation to resistant mutants was ≥1%.

Note: The isolation of Smd clones is achieved by use of Sm sensitive wild type strains as the starting material. Strains preferably have a low MIC value.

(b) Practice-Orientated Isolation of Smd Mutants of Campylobacter

Bacterial material obtained from a Caso agar Petri dish culture (24 h/39° C.; about 10¹⁰ cfu) that had been inoculated in such a way that the entire surface of the disc was covered was plated on 1 or 2 Caso agar Petri dishes supplemented with 100 μg streptomycin/ml and incubated for 72 h at 39° C. Depending on the strain≤10 colonies/plate (average value) having normal sizes and colonies having slightly decreased sizes (streptomycin resistant and MD Sm “res” clones) were detectable. In addition, colonies having a clearly reduced size (diameter is ≤25% of the normal size) with a frequency of about 20%—compared to the colonies having normal sizes and colonies having slightly reduced sizes—could be detected. About one-third of these colonies were Smd clones.

The calculated frequency of the Smd clones in relation to resistant mutants was ≥5%.

EXAMPLE 3 Selection and Isolation of Sm-id Mutants

(a) Isolation of Salmonella Sm-id Mutants from Smd Clones

About 10⁹ cfu (per petri dish) of a washed Smd mutant were plated with SC medium and incubated for 48 h at 37° C. From the attenuated revertants obtained only such mutants were further treated that showed a colony size of about ≥50% compared to the wild type strain colonies (according to the objective to obtain Sm-id clones having only low attenuation).

(b) Isolation of Campylobacter Sm-id Mutants from Smd Clones

Bacterial material obtained from a Caso agar (supplemented with 100 μg streptomycin/ml) Petri dish culture (24 h/39° C.) that had been inoculated in such a way that the entire surface of the disc was covered was subjected to one washing step, plated on Caso medium in a ratio of 1:1 (about 3×10⁹ cfu) to 1:4 and then incubated for 72 h at 39° C. Under these culturing conditions the majority of Smd clones showed the development of ≤10 attenuated revertants (on average). Most of these attenuated revertants were Sm sensitive. Generally, Sm-id clones showing a reduced colony size of about ≥50% compared to the wild type strain colonies were further processed.

Some strains, e.g., Campylobacter jejuni, allowed isolation only from Sm-id having a colony size of ≤50% compared to the wild type strain.

Note: Not all Campylobacter strains and the Smd mutants derived thereof allow isolation of Sm-id revertants without any problems. However, it is possible to isolate Sm-id revertants also from the problematic strains using, for example, several independent Smd mutants.

EXAMPLE 4 Isolation of an Additional MD Antibiotic “res” Mutant

The incorporation of an additional MD antibiotic “res” mutation in selected Sm-id mutants as third marker for attenuation and recognition was carried out as already described above.

Briefly,

(a) Salmonella: 10⁹⁻¹⁰ cfu of the selected Sm-id clones were incubated on SC medium supplemented with an about tenfold MIC value concentration of rifampicin or streptomycin (as regards fusidine acid the about fourfold MIC value concentration), respectively, and incubated for 48 hours at 37° C.

(b) Campylobacter: The material of a Petri dish culture (Caso medium) that was inoculated with the Sm-id mutant in such a way that it covered the whole surface and incubated for 24 h at 39° C. was plated at a ratio of 1:4 to 1:8 on Caso medium supplemented with 200 μg fosomycin/ml or 100 μg streptomycin/ml and incubated for ≥72 h at 39° C.

The colonies showing (more or less) reduced sizes were isolated and subjected to serial passages. About 20% of these clones maintained the clone specifically graded reduction of colony size as a stabile feature.

EXAMPLE 5 Generation of Vaccine Strains Having 4 or 6 Attenuated Mutations

The generation of vaccine strains having 4 or 6 attenuated mutations was achieved by sequentially incorporating a second and, optionally, a third Sm-id suppressor mutation into a basic Sm-id I clone: Sm-id I/Sm-id II/Sm-id III.

{a) Salmonella: About 10¹⁰ cfu of the basic Sm-id I mutant (or the Sm-id II starting strain) were plated on SC medium supplemented with 500 μg streptomycin/ml and incubated for 48 h at 37° C. About 5% of Sm-resistant colonies are Smd mutants (now growing primarily as colonies having “normal sizes”). By use of these Smd clones derived from Sm-id I strains and Sm-id II strains, respectively, Sm-id mutants were again isolated according to the approach described in Example 3a. Clones having the desired reduction of colony size were treated further.

(b) Campylobacter: The material obtained from a Caso medium Petri dish culture that was inoculated with an Sm-id mutant in such a way that the entire surface was covered and incubated for 24 h at 39° C. was plated at a ratio of 1:4 on Caso medium supplemented with 100 μg streptomycin/ml and incubated for 72 h at 39° C. Besides the about 15 Sm resistant colonies having a “normal size” 2 to 3 small colonies could be detected. 50% of these colonies are Smd clones. These Smd clones (derived from Sm-id I strains and Sm-id II strains, respectively) were used as starting clones—according to Example 3{b)—for again isolating Sm-id mutants. Clones showing the desired reduction of colony size were treated further.

EXAMPLE 6 Isolation of an MD Antibiotic “res” Mutant from Selected Sm-id II Mutants

(a) Salmonella: The incorporation of an advantageous MD antibiotic “res” mutation into selected Sm-id I/Sm-id II mutants as an additional 5^(th) attenuation- and recognition marker was carried out analogously according to the approach described in Example 4(a).

(b) Campylobacter: The incorporation of an advantageous MD antibiotic “res” mutation into selected Sm-id I/Sm-id II mutants as an additional 5^(th) attenuation- and recognition marker was carried oat analogously according to the approach described in Example 4(b).

Note: The approach described above can also be used for the additional incorporation of an MD antibiotic “res” mutation into selected Sm-id III mutants (having six attenuated mutations) as 7^(th) marker for attenuation and recognition. However, this might result in over-attenuation, which might interfere with relevancy to practice.

EXAMPLE 7 Colony Sizes Converted to Bar Graphs for Prospectively Oriented Evaluation of the Probable Degree of Attenuation

Suspensions of the corresponding wild type strains and the MD mutants derived from these strains are diluted logarithmically and then plated on culture medium in such a way that per Petri dish 10 to 50 well definable single colonies can be obtained. At least 5 Petri dishes per grade of dilution are prepared in order to compensate for differences in growth due to the medium. Single colonies grown under standardized conditions (e.g., identical times of incubation, identical layer thicknesses of the medium) are photographed. Digital photographs are processed with the CellProfiler program (Broad Institute): The diameters of the individual colonies were determined and saved. After averaging of the values the data are plotted as bar graphs in relation to the sizes of the wild type strain colonies (given as 100%).

EXAMPLE 8 Preparation of Vaccines from Suitable Vaccine Strains and Use for Vaccination of Chicks/Chicken and Further Hosts to be Protected, Respectively

For the preparation of live vaccines vaccine strains harbouring three (four, five and six, respectively) attenuating mutations were grown in common liquid media up to logarithmic phase. Vaccine suspensions and vaccine sediments, respectively, were supplemented with a suitable stabilisator and subsequently lyophilized. The vaccines obtained were administrated (according to the kind of indication one, two or three doses) by oral or parenteral administration. 

What is claimed is:
 1. A stable attenuated bacterial strain comprising three to seven attenuating metabolic drift (MD) mutations; wherein at a first attenuating MD mutation is marked by dependence of growth on a first antibiotic and the strain bearing the first attenuating MD mutation grows as a mini colony in the presence of the first antibiotic; wherein a second attenuating MD mutation is marked by reversion to independence of growth in the absence of the first antibiotic; and wherein a third attenuating MD mutation is marked by resistance to a second antibiotic; wherein the bacterial strain with independence of growth in the absence of the first antibiotic grows in a colony having a reduced size at least 50% as compared to a colony of a corresponding starting natural bacterial strain; wherein the bacterial strain resistant to the second antibiotic grows in a colony of reduced size but greater than 25% in size as compared to the corresponding starting natural bacterial strain; wherein said growth of bacterial strain resistant to the second antibiotic in a colony of said reduced size is a stable phenotype of the bacterial strain carrying the three to seven attenuating mutations; wherein the first antibiotic is selected from the group consisting of streptomycin, neomycin, kanamycin, spectinomycin, gentamicin, amikacin, tobramycin, rifampicin, fusidic acid, and nalidixic acid; wherein the second antibiotic is selected from the group consisting of streptomycin, neomycin, kanamycin, spectinomycin, gentamicin, amikacin, tobramycin, rifampicin, fusidic acid, nalidixic acid, and fosfomycin; and wherein the bacterial strain is selected from the group consisting of Aeromonas species (sp.), Bacillus cereus, Bordetella sp., Campylobacter species (sp.), Escherichia coli, Klebsiella sp., Listeria sp., Ornithobacterium rhinotracheale, Pasteurella/Avibacterium sp., Pseudomonas sp., Riemerella sp., Salmonella sp., Shigella sp., Staphylococcus aureus, and Yersinia sp.; wherein a colony of the attenuated bacterial strain having a size of <10% as compared to a colony of the corresponding starting natural bacterial strain defines a mini colony; wherein the colony size is determined by the diameter of the colony; and wherein the stable attenuated bacterial strain is not over-attenuated.
 2. A composition comprising the stable attenuated bacterial strain of claim 1 and a biologically acceptable carrier; wherein the stable attenuated bacterial strain is Salmonella or Campylobacter.
 3. The composition of claim 2 which is freeze-dried.
 4. A stable attenuated bacterial strain comprising five to seven attenuating metabolic drift (MD) mutations; wherein a first attenuating MD mutation is marked by dependence of growth on a first antibiotic and the strain bearing the first attenuating MD mutation grows as a mini colony in the presence of the first antibiotic; wherein a second attenuating MD mutation is marked by reversion to independence of growth in the absence of the first antibiotic; and wherein a third attenuating MD mutation is marked by dependence of growth on a second antibiotic and the strain bearing the third attenuating MD mutation grows as a mini colony in the presence of the second antibiotic; wherein a fourth attenuating MD mutation is marked by reversion to a third antibiotic; wherein the bacterial strain with independence of growth in the absence of the first antibiotic and the second antibiotic grows in a colony having a reduced size at least 50% as compared to a colony of the corresponding starting natural bacterial strain; wherein the bacterial strain resistant to the third antibiotic grows in a colony of reduced size but greater than 25% in size as compared to the corresponding starting natural bacterial strain; wherein said growth of bacterial strain resistant to the third antibiotic in a colony of said reduced size is a stable phenotype of the bacterial strain carrying the five to seven attenuating mutations; wherein the bacterial strain is selected from the group consisting of Aeromonas species (sp.), Bacillus cereus, Bordetella sp., Campylobacter species (sp.), Escherichia coli, Klebsiella sp., Listeria sp., Ornithobacterium rhinotracheale, Pasteurella/Avibacterium sp., Pseudomonas sp., Riemerella sp., Salmonella sp., Shigella sp., Staphylococcus aureus, and Yersinia sp.; wherein the first antibiotic is selected from the group consisting of streptomycin, neomycin, kanamycin, spectinomycin, gentamicin, amikacin, tobramycin, rifampicin, fusidic acid, and nalidixic acid; wherein the second antibiotic is selected from the group consisting of streptomycin, neomycin, kanamycin, spectinomycin, gentamicin, amikacin, tobramycin, rifampicin, fusidic acid, nalidixic acid, and fosfomycin; wherein the third antibiotic is selected from the group consisting of streptomycin, neomycin, kanamycin, spectinomycin, gentamicin, amikacin, tobramycin, rifampicin, fusidic acid, nalidixic acid, and fosfomycin; wherein a colony of the attenuated bacterial strain having a size of <10% as compared to a colony of the corresponding starting natural bacterial strain defines a mini colony; wherein the colony size is determined by the diameter of the colony; and wherein the stable attenuated bacterial strain is not over-attenuated.
 5. A composition comprising the stable attenuated bacterial strain of claim 4 and a biologically acceptable carrier; wherein the stable attenuated bacterial strain is Salmonella or Campylobacter.
 6. The composition of claim 5 which is freeze-dried.
 7. The stable attenuated bacterial strain of claim 1, wherein the first antibiotic is streptomycin; and wherein the second antibiotic is rifampicin.
 8. The stable attenuated bacterial strain of claim 4, wherein one of the first antibiotic, the second antibiotic, and the third antibiotic is streptomycin. 